CN112175090B - 一种人溶菌酶结合物及其制备方法和应用 - Google Patents
一种人溶菌酶结合物及其制备方法和应用 Download PDFInfo
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- CN112175090B CN112175090B CN201910594781.4A CN201910594781A CN112175090B CN 112175090 B CN112175090 B CN 112175090B CN 201910594781 A CN201910594781 A CN 201910594781A CN 112175090 B CN112175090 B CN 112175090B
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- human lysozyme
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Abstract
本申请涉及生物药品技术领域,具体公开了一种人溶菌酶结合物及其制备方法和应用。基因重组人溶菌酶克服了现有溶菌酶对人体有免疫排斥反应而只能外用的缺陷。本发明将人溶菌酶与抗病毒小分子抗体以及其它抗体偶联制成人溶菌酶‑抗体结合物,增加溶菌酶特异性抗病毒能力,赋予了溶菌酶新的药理作用;同时利用人溶菌酶特有的激活血小板、改善组织局部循环、分解脓液的作用,弥补了抗体在消除炎症和溃疡方面的不足;本发明还提供人溶菌酶以及抗病毒人溶菌酶‑抗体结合物制成的液晶微囊、凝胶、栓剂、乳膏剂、洗液、片剂、泡腾片、胶囊剂、软胶囊、喷雾剂及注射剂,在防治细菌和病毒感染性疾病的药物、消毒产品、保健品和医疗器械、畜牧业方面的应用。
Description
技术领域
本发明涉及生物药品技术领域,尤其涉及一种新型的人溶菌酶结合物及其制备方法和应用。
背景技术
溶菌酶是一种无毒、无害、无耐药性的高盐基水解蛋白酶,且具有一定的保健作用;其中人溶菌酶是人体内的一种蛋白质,和人体天然兼容,比其他溶菌酶更安全,没有任何刺激性和副作用;同时,人溶菌酶比其它溶菌酶具有更高的抗病毒能力和溶菌活性以及热稳定性。但是,人溶菌酶存在提取困难和活性难以保证以及来源受限、产量极低、质量不稳定、含有未知病原、价格高昂等问题。现国内生产的溶菌酶制剂主要是从鸡蛋的蛋壳和蛋清中提取得来的,国外则以动物和霉菌为原料进行生产。无论是从蛋壳和蛋清提取的溶菌酶,还是从动物和霉菌中生产的溶菌酶都是一种异源性蛋白,对人体有免疫排斥反应,也无法对抗机体内的细菌、病毒等,而且其活性也较低,热稳定性也差。
另外,溶菌酶虽然有抑菌作用,但是,不具有特异性抗病毒的功能,这就限制了它在医药和保健方面的应用。
发明内容
本发明一方面提供一种基因重组人溶菌酶、基因重组抗病毒人溶菌酶-抗体结合物及其组合物以及制备方法和应用,克服现有技术生产的溶菌酶之缺陷,一方面由于基因重组人溶菌酶与人体具有天然兼容性,解决了异源性蛋白的免疫排斥反应问题,临床应用时,比其他溶菌酶更安全并延长疗效,特别是可制成注射剂代替抗生素用于治疗感染性疾病;另一方面基因重组人溶菌酶比普通溶菌酶抗病毒功能更强并可产生提升免疫力、激活血小板,改善组织局部循环障碍,分解脓液的独特作用。
本发明的另一方面,提供一种人溶菌酶与抗病毒小分子抗体或IgY、IgG、单抗、双特异性抗体、羊鸵抗体等抗体偶联制成的人溶菌酶-抗体结合物,增加了溶菌酶特异性抗病毒能力,赋予了溶菌酶新的药理作用。
本发明解决技术问题所采用的技术方案是:提供一种人溶菌酶结合物,包括相互偶联的人溶菌酶与抗体,所述人溶菌酶为基因重组表达的产物。
所述基因重组人溶菌酶由如下方法制备得到:
本发明将人溶菌酶基因克隆到原核和真核生物中进行表达。
人溶菌酶HLY的氨基酸序列如下所示:Lys-Val-Phe-Glu-Arg-Cys-Clu-Leu-Ala-Arg-Thr-Leu-Lys-Arg-Leu-Gly-Met-Asp-Gly-Tyr-Arg-Gly-Ile-Ser-Leu-Ala-Asn-Trp-Met-Cys-Leu-Ala-Lys-Trp-Glu-Ser-Gly-Tyr-Asn-Thr-Arg-Ala-Thr-Asn-Tyr-Asn-Ala-Gly-Asp-Arg-Ser-Thr-Asp-Tyr-Gly-Ile-Phe-Gln-Ile-Asn-Ser-Arg-Tyr-Trp-Cys-Asn-Asp-Gly-Lys-Thr-Pro-Gly-Ala-Val-Asn-Ala-Cys-His-Leu-Ser-Cys-Ser-Ala-Leu-Leu-Gln-Asp-Asn-Ile-Ala-Asp-Ala-Val-Ala-Cys-Ala-Lys-Arg-Val-Val-Arg-Asp-Pro-Gln-Gly-Ile-Arg-Ala-Trp-Val-Ala-Trp-Arg-Asn-Arg-Cys-Gln-Asn-Arg-Asp-Val-Arg-Gln-Tyr-Val-Gln-Gly-Cys-Gly-Val
一、以大肠杆菌等作宿主的原核表达系统表达人溶菌酶基因生产重组人溶菌酶
构建含有人溶菌酶基因的E.coli工程菌JPB-HLY,采用化学合成人溶菌酶基因和温控串联启动子PRPL,经培养、诱导后,所得菌体破碎,酶蛋白经变性、复性处理后所得粗酶液经Express-Ion S阳离子交换柱层析,得到了电泳纯的人溶菌酶。
二、以枯草芽孢杆菌和链霉菌等作宿主的原核表达系统表达人溶菌酶基因生产重组人溶菌酶
利用回复PcR(reucrsivePCR)合成人溶菌酶cDNA,再克隆到表达载体PlGF中,接着转染枯草芽孢杆菌或黑曲霉或其它霉菌,在界乙烯毗咯烷酮P(钾)完全培养基中培养,酶蛋白经变性、复性处理后所得粗酶液经Express-Ion S阳离子交换柱层析,得到了电泳纯的人溶菌酶。
三、以巴斯德毕赤酵母等酵母菌作宿主表达人溶菌酶基因生产重组人溶菌酶
酵母作为一种表达外源基因的宿主菌,既具有操作简单,生长快等特点,又具有真核细胞翻译后修饰加工系统;可以大规模生产,从而降低成本。常用的酵母表达系统有酿酒酵母表达系统,甲基营养型酵母表达系统和裂殖酵母表达系统。采用其中的毕赤酵母表达人溶菌酶,分为分泌型表达和胞内表达两种。下面分别加以说明:
(一)分泌型表达
从人胎盘组织中提取人溶菌酶编码基因,切除基因信号肽,将其连接至毕赤酵母分泌表达载体pPICZαA上,电转化至酵母菌株X-33感受态中,构建重组分泌型酵母菌株,甲醇诱导表达48h。通过定点突变技术将表达载体pPICZαA信号肽C端与hLYZ成熟肽N端相连的2个氨基酸Leu和Glu分别突变成Pro和His,甲醇诱导表达72h,可提高人溶菌酶的表达量。
将发酵液离心,收集上清液,用6k~30k的中空纤维膜截留。收集的蛋白溶液先流过SPFF强阳离子交换层析柱,洗脱收集的蛋白液再流过DEAE弱阴离子交换层析柱,得到纯的人溶菌酶。
(二)胞内表达
将来自胎盘的人溶菌酶基因hLYZ克隆至表达载体pPICZA上,将经验证后的重组载体电转化至毕赤酵母X-33中,通过抗生素浓度梯度筛选出高拷贝转化子,进行甲醇诱导表达。通过单因素和正交试验确定培养基最优配方和最优的发酵条件,达到优化目的。将发酵液离心,收集上清液,用6k~30k的中空纤维膜截留。收集的蛋白溶液先流过SPFF强阳离子交换层析柱,洗脱收集的蛋白液再流过DEAE弱阴离子交换层析柱,得到纯的人溶菌酶。
以上用原核和真核表达系统表达人溶菌酶基因生产重组人溶菌酶的三种方法为例来进行说明,实际应用不限于所列的这些方法,这里就不赘述。
本发明解决技术问题所采用的另一个技术方案是:提供一种人溶菌酶结合物的制备方法,包括如下步骤:
S1、体外表达人溶菌酶:将人溶菌酶基因克隆到表达系统,表达人溶菌酶基因以生产重组人溶菌酶;
S2、连接抗体:将所述人溶菌酶与抗体偶联,得到所述人溶菌酶结合物。
一种人溶菌酶与抗病毒小分子抗体或IgY、IgG、单抗、双特异性抗体、羊鸵抗体等抗体偶联制成的抗病毒人溶菌酶-抗体结合物,所述基因重组人溶菌酶与抗病毒小分子抗体或IgY、IgG、单抗、双特异性抗体、羊鸵抗体等抗体偶联制成的抗病毒人溶菌酶-抗体结合物采用如下方法制备得到:
将人溶菌酶连接到抗病毒小分子抗体或其它抗体上。其中的抗病毒小分子抗体或其它抗体可以是抗HPV-E6/E7-Fab和抗HPV-E6/E7-IgY,也可以是抗广谱HPV-Fab和抗广谱HPV-IgY,也可以是其它抗病毒IgY及其小分子抗体,如抗流感病毒IgY及其小分子抗体、抗咽喉炎致病菌IgY及其小分子抗体、抗疱疹病毒IgY及其小分子抗体、抗幽门杆菌IgY及其小分子抗体、抗妇科炎症致病菌IgY及其小分子抗体、抗口腔疾病致病菌(包括变链菌和牙周炎致病菌)IgY及其小分子抗体、抗痤疮致病菌IgY及其小分子抗体,也可以是IgG、单抗、双特异性抗体、羊鸵抗体等抗体。
本发明通过一个化学链接或者基因重组技术将人溶菌酶连接到抗病毒小分子抗体或IgY、IgG、单抗、双特异性抗体、羊鸵抗体等抗体上。具体化学链接的方法有很多种,如碳化二亚胺法、戊二醛法、混和酸酐法、过碘酸氧化法、琥珀酸酐法、羧甲基羟胺法、重氮化的对氨基苯甲酸法、一氯醋酸钠法等,其中最主要的有两种:活性酯法和酸酐法。也可采用SPDP等异型双功能试剂作为交联剂进行偶联。具体过程,这里就不赘述。
本发明以异型双功能试剂作交联剂进行偶联的方法为例说明,实际应用中,不限于这个方法,可采用其它任何同型双功能试剂或异型双功能试剂作为交联剂进行偶联。
具体操作说明如下:
(一)用N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯处理人溶菌酶:
将人溶菌酶溶于缓冲液中,得到人溶菌酶溶液;再用无水乙醇溶解N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯,然后加入人溶菌酶溶液中,在23~25℃反应30分钟。将反应液通过SephadexG-25柱以除去多余的SPDP及副产物,收集并浓缩人溶菌酶蛋白液(标记为HRP-PDP)。
(二)用N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯处理抗病毒小分子抗体或其它抗体:
将抗病毒小分子抗体或其它抗体溶于缓冲液中,得到抗体溶液;再用无水乙醇溶解N-琥珀酰亚胺基3-(2-吡啶基二硫)丙酸酯,然后加入抗体溶液中,在23~25℃反应30分钟。将反应液通过SephadexG-25柱以除去多余的SPDP及副产物,收集并浓缩抗体蛋白液(标记为ANTI-PDP)。
(三)人溶菌酶蛋白的还原:取上述人溶菌酶蛋白液(标记为HRP-PDP),加入固体二巯基苏糖醇(DTT),在23—25℃反应约25分钟。然后,过Sephadex G-25柱,收集并浓缩还原的人溶菌酶蛋白液(标记为HRP-SH)。
(四)人溶菌酶和抗体的偶联:将上述制得的还原的人溶菌酶蛋白液(标记为HRP-SH)和抗体蛋白液(标记为ANTI-PDP)混合,在4℃左右温度下反应约20小时,然后浓缩,获得人溶菌酶和抗体结合物。
将人溶菌酶与抗病毒小分子抗体或IgY、IgG、单抗、双特异性抗体、羊鸵抗体等抗体偶联制成人溶菌酶-抗体结合物可得到两大有益效果:
一、使人溶菌酶具有特异性抗病毒功效。
二、人溶菌酶特有的激活血小板、改善组织局部循环障碍、分解脓液的作用可弥补抗体在消除炎症和溃疡方面的不足。
这种结合物可简称为“抗体溶菌酶”。
本发明还提供了上述的基因重组人溶菌酶以及抗病毒人溶菌酶-抗体结合物作为制备用于防治人乳头瘤病毒(HPV)等病毒感染、和/或用于预防和治疗感染性疾病的药物、消毒产品、保健品或医疗器械中的应用。是抗生素和化学药的最佳替代。基于基因重组人溶菌酶与人体天然兼容的特质,可制成直接注射到人体内治疗疾病的水针剂和粉针剂。
本发明还提供了一种组合物,包括了上述的基因重组人溶菌酶、基因重组抗病毒人溶菌酶-抗体结合物以及至少一种其它药学上可接受的组分。
在本发明的组合物中,所述基因重组人溶菌酶以及基因重组抗病毒人溶菌酶-抗体结合物添加辅料或基料或者化学药、中药、生物制品或生物药,制成液晶微囊、凝胶、栓剂、乳膏剂、洗液、片剂、泡腾片、胶囊剂、软胶囊、喷雾剂中的至少一种。
本发明还提供了上述的组合物作为制备用于防治人乳头瘤病毒(HPV)等病毒感染、和/或用于预防和治疗感染性疾病的药物、消毒产品、保健品或医疗器械中的应用。
基因重组人溶菌酶、基因重组抗病毒人溶菌酶-抗体结合物以及其组合物可制成各种稳定制剂。所述制剂包括但不限于这些制剂:
优选地,该制剂还包括赋形剂、填充剂、溶剂、助溶剂、表面活性剂和胶囊辅料中一种或多种。
优选地,该制剂为阴道凝胶、栓剂、洗液等。
优选地,该制剂为片剂、喷雾剂、粉剂、口服液剂或胶囊。
优选地,基因重组人溶菌酶注射剂为水针剂和粉针剂中的至少一种。
所述基因重组人溶菌酶、基因重组抗病毒人溶菌酶-抗体结合物以及其组合物添加辅料或基料或者化学药、中药、生物制品或生物药,制成液晶微囊、脂质体液晶微囊、凝胶、栓剂、乳膏剂、洗液、片剂、泡腾片、胶囊剂、软胶囊、喷雾剂中的至少一种,或者將基因重组人溶菌酶制成注射用水针剂和粉针剂中的至少一种,作为一种预防和治疗细菌和病毒感染的生物药。
实施本发明,具有如下有益效果:本发明通过将溶菌酶与抗体结合的方式,弥补了溶菌酶的不足,增加其功能,同时解决人溶菌酶大规模生产的问题,扩大溶菌酶在疾病防治、公共卫生、食品安全以及畜牧业领域的应用。
具体实施方式
下面结合具体实施例对本发明的技术方案进行详细的说明,但是所述实施例的说明,仅仅是本发明的一部分实施例,其中大部分并不仅限于此。
下面实施例以采用基因重组人溶菌酶、基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物以及基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物为功效成份制成各种制剂的方法为例进行说明,实际应用中不限于基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物,也不限于基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物,可采用基因重组人溶菌酶与其它任何抗体偶联的结合物为功效成份制成各种制剂,这里就不赘述。
实施例1:基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物凝胶使HPV检测为阳性的妇女“阳转阴”试验
(1)试验对象:年龄21~45岁,无重大脏器疾病者,无药物过敏史和无过敏体质者),有性生活史的妇女,经HPV抗体检测和液基细胞学检查,确诊为为阳性的患者。共120例。随机分为治疗组和对照组各60例。
(2)试验方法
治疗组:采用基因重组人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物妇科凝胶进行治疗。每天先用基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物洗液清洗,然后阴道灌注基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物妇科凝胶,每天一次,每次使用一支,每个疗程连续使用三盒共9支。连续三个疗程。按要求接受全程追踪随访观察,患者入组35天后复诊,观察HPV感染转阴情况。
对照组:采用普通妇科凝胶进行治疗。每天阴道灌注普通妇科凝胶,每天一次,每次使用一支,每个疗程连续使用三盒共9支。连续三个疗程。按要求接受全程追踪随访观察,患者入组35天后复诊,观察HPV感染转阴情况。
(3)追踪随访
患者按要求入组后,接受口头知情并详细填写各项临床观察表,第35天作相应检查,并将结果详细填入观察表中。如果在治疗过程中出现药物不良反应要详细填入表中,出现严重反应,立即上报相应部门并及时处理。
(4)治疗疗效判定标准
HPV检测结果分:(1)HPV浓度基本不变,(2)HPV检测完全阴性。无效为:HPV检测为HPV浓度基本不变,结果维持阳性。
(5)结果
试验35天后复诊,治疗组复查,HPV浓度显著下降转阴51例,无效9例,转阴率为85%。对照组复查HPV转阴12例,转阴率为20%;无效48例。P<0.05,二者比较有显著差异。
实施例2:制备基因重组人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物脂质体液晶微囊。
具体实施步骤如下:
(1)把卵磷脂和胆固醇各50%按1:5比例溶于乙醚中;
(2)将所制得的基因重组人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物干粉按1:100比例加入4mmol/L磷酸盐缓冲液(PBS)配成浓度1%的抗体Fab溶液;
(3)将卵磷脂和胆固醇乙醚溶液和基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物的PBS溶液按3:1比例混合均匀;
(4)超声处理2min(每处理0.5min,间歇.0.5min);
(5)在水浴中减压旋转蒸发至呈凝胶状,漩涡振荡使凝胶转相,再继续蒸发除尽乙醚;
(6)超速离心(35000r/min,30min)分离除去未包入的抗体Fab;
(7)沉淀用水洗二次,置入高速离心机离心,得沉淀;
(8)将所得的沉淀用10mmol/L PBS稀释,即制得基因重组人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物脂质体液晶微囊。
实施例3:生产基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物凝胶
配方:
工艺:
(1)将配方量乳糖、羟乙基纤维素(HEC)、薄荷油、香精用紫外光照射灭菌消毒24小时,无菌密封备用;
(2)按配方量蒸馏水加热至至90℃,再分别加入S-40和K-30分散剂以及乳糖,搅拌30分钟以上,溶解均匀;再冷却至60℃,分别缓慢加入吐温(滴加)和薄荷油以及甘油(滴加),低速揽拌60分钟至充分溶解;保持温度在60℃,形成溶液A;
(3)将溶液A加热至60℃,保持温度在60℃,一边搅拌一边将医药级尿囊素加入溶液A中,低速搅拌60min,形成溶液B;
(4)将配方量表皮生长因子用适量95%的酒精溶解,一边搅拌一边加热至60℃,直至完全溶解;保持温度在60℃,得表皮生长因子酒精溶液C;
(5)一边搅拌一边将溶液C滴加入溶液B中,继续高速搅拌60min,温度始终控制在50-60℃,直至完全溶解,然后冷却至室温,得溶液D;
(6)一边搅拌一边将基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物加入溶液D中,低速搅拌60min,直至完全混合均匀,得溶液E;
(7)一边搅拌一边将香精(玫瑰花香精)加入溶液E中,低速搅拌60min,直至完全溶解,得溶液F;
(8)一边搅拌一边将配方量羟乙基纤维素(HEC)加入溶液F中,低速搅拌60min,直至完全溶解,形成均质稳定的溶液,测试溶液粘稠度,适合后,得溶液G;
(9)用pH计测量溶液G的pH值,用柠檬酸调节pH值至4.5±0.1;
(10)静置一夜,直至上部泡沫全部消溶后,再将溶液G分装在清洗消毒过的塑料桶中,贴上标签出厂。
实施例4:生产基因重组人溶菌酶-抗多种人乳头瘤病毒(HPV16、HPV18、HPV31、HPV58)E6/E7小分子抗体Fab结合物乳膏剂
配方:
工艺:
(1)按配方量蒸馏水加热至至90℃,再分别加入Span-80和吐温K-30以及乳糖,搅拌30分钟以上,溶解均匀;再冷却至60℃,分别缓慢加入山梨酸钾和甘油(滴加),低速揽拌60分钟至充分溶解;保持温度在60℃,形成溶液A;
(2)将配方量薄荷醇用适量95%的酒精溶解,一边搅拌一边加热至60℃,直至完全溶解;得溶液B;
(3)将溶液A加热至60℃,保持温度在60℃,一边搅拌一边将溶液B滴加入溶液A,低速揽拌30分钟至充分溶解;保持温度在60℃,形成溶液C;
(4)将液体石蜡加热至60℃,保持温度在60℃,一边低速搅拌一边分别将凡士林、十八烷酸、十八烷醇加入,一边低速搅拌一边加热使其熔融,形成溶液D;
(5)将溶液D加热至60℃,保持温度在60℃,一边搅拌一边将溶液C加入溶液D中,低速搅拌60min,形成溶液E;
(6)降低搅拌速度,乳化一定时间,成型后停止加热,冷却至40℃左右,然后一边低速搅拌一边将基因重组人溶菌酶-抗多种人乳头瘤病毒(HPV16、HPV18、HPV31、HPV58)E6/E7小分子抗体Fab结合物加入溶液E中,充分研磨搅拌均匀,制成o/w乳膏,静置冷却至室温,得溶液F;
(7)用pH计测量溶液F的pH值,调节pH值至4.5±0.1;
(8)静置一夜,将溶液F分装在清洗消毒过的塑料管中,贴上标签装入包装盒出厂。
实施例5:生产基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物阴道栓剂
配方:
工艺:
(1)按配方量90%蒸馏水加热至至90℃,加入明胶溶胀。冷却至60℃,形成溶液A;
(2)保持溶液A温度在60℃,一边搅拌一边将配方量甘油加入溶液A,混匀,冷至40℃以下,得溶液B;
(3)将基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物加入溶液B,低速揽拌30分钟至充分溶解;冷却至室温,得溶液C;
(4)用pH计测量溶液C的pH值,调节pH值至4.5±0.1;
(5)静置一夜,将溶液C分装在清洗消毒过的塑料管中,贴上标签装入包装盒出厂。
实施例6:生产基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物洗液
配方:
工艺:
(1)将配方量海藻精华、医药级甘油、薄荷油、香精以及403、503发泡剂用紫外光照射灭菌消毒24小时,无菌密封备用;
(2)将配方量蒸馏水加热至90℃,停留15分钟;再冷却至60℃,一边搅拌一边先后加入海藻精华和医药级甘油,低速揽拌30分钟,直至完全溶解,冷却至室温,得溶液A;
(3)一边搅拌一边将香精(玫瑰花香精)加入溶液A中,低速搅拌60min,直至完全溶解,得溶液B;
(4)将403加热至80℃,然后一边搅拌一边将503徐徐滴加入到403中,低速搅拌30min,得到溶液C;
(5)保持溶液C温度在80℃,一边搅拌一边将薄荷油徐徐滴加入到溶液C中,低速搅拌60min,得到溶液D;
(6)将溶液D加热至90℃高温灭菌5min,然后冷却至室温;
(7)一边搅拌一边将溶液B缓慢地加到溶液D中,低速搅拌60min;如未形成均质稳定的乳状溶液,则需延长搅拌时间,制得溶液E;
(8)一边搅拌一边将基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物缓慢地加到溶液E中,低速搅拌60min;如未形成均质稳定的乳状溶液,则需延长搅拌时间,制得溶液F;
(9)用pH计测量溶液F的pH值,采用柠檬酸或pH4.5磷酸氢二钠–柠檬酸缓冲液调节pH值至4.5±0.1;
(10)静置一夜,直至上部泡沫全部消溶后,再将溶液F液分装在清洗消毒过的塑料容器中,贴上标签出厂。
实施例7:生产基因重组人溶菌酶-抗多种人乳头瘤病毒(HPV16、HPV18、HPV31、HPV58)E6/E7小分子抗体Fab结合物泡腾片
配方:
工艺:
(1)将NaHCO360℃干燥2h,加20%PEG乙醇液适量制软材,14目尼龙筛制粒60℃干燥备用;
(2)枸椽酸、乳糖、MCC混匀80目粉碎,加于2%HPMC醇液中搅匀;
(3)加入基因重组人溶菌酶-抗多种人乳头瘤病毒(HPV16、HPV18、HPV31、HPV58)E6/E7小分子抗体Fab结合物、氢氧化铝混匀过一次14目筛,再加硬脂酸镁混匀压片即得;
(4)检验合格后水泡眼包装,外套铝塑复合膜塑料袋密封,装盒,装箱入库。
实施例8:生产基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物片剂
配方:
工艺
(1)山梨糖醇过60目筛两次备用;
(2)羧甲基纤维素分散于30%乙醇中制成1%乙醇溶液;
(3)将(1)项用适量(2)项制软材,14目筛网制粒,60℃通风干燥,18目筛整粒;用40目筛筛出适量细粉与基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物充分混匀;
(4)再拌入硬脂酸镁,一起与整批颗粒混合均匀,密闭4小时以上;
(5)压片机压片,每片600mg;
(6)检验合格后,包装,全验出厂。
实施例9:生产基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物硬胶囊。
配方:
工艺:
(1)将基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物和药用葡萄糖按重量比1:1比例混合,充分搅拌均匀;
(2)用胶囊充填机将基因重组抗病毒人溶菌酶-抗广谱HPV-Fab小分子抗体结合物和药用葡萄糖混合物定量分装入药用胶囊,每粒装300mg;
(3)将胶囊装瓶,检验出厂。
实施例10:生产基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物软胶囊
配方:
工艺:
(1)将吐温加热至50℃,一边搅拌一边将大豆油徐徐滴加入到吐温中,高速(3000r/min)搅拌15min,冷却到室温,得到溶液A;
(2)将配方量蒸馏水采用高压蒸气灭菌,加热到121-126℃,压力升至102.9kPa(1.05kg/cm2),停留60min,然后冷却到室温;
(3)将配方量基因重组人溶菌酶-抗广谱HPV-Fab小分子抗体结合物先用高温灭菌后冷却到室温的蒸馏水溶解,充分搅拌均匀,制成人溶菌酶-抗体水溶液B;
(4)一边搅拌一边缓慢地将基因重组抗病毒人溶菌酶-抗广谱HPV-Fab小分子抗体结合物水溶液B加入到溶液A中,高速(3000r/min)搅拌60min以上,得到溶液C;经检测,溶液C中80%粒径在2~50μm范围,平均收率可达91.4%;
(5)将所制成的纳米微囊溶液C加入软胶囊机滚压模切断成型,每粒300mg;
(6)将压模成型的软胶囊干燥定型;
(7)采用铝塑包装机包装;
(8)纸盒包装,检验合格出厂。
实施例11:生产基因重组人溶菌酶注射剂
将所制得的基因重组人溶菌酶制成一种没有副作用和耐药性的新型注射剂。具体配方和生产工艺如下:
配方:
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工艺:
(1)将配方量注射用水加0.1%针用活性炭,60℃搅拌15min;脱炭过滤;滤液密闭加热100℃30min,冷却至室温备用;
(2)一边搅拌一边分别将配方量磷酸二氢钠一水合物和磷酸氢二钠七水合物以及氯化钠加入占配方量90%的经活性炭处理过的灭菌注射用水中,加热溶解;另取聚山梨醇酯-80,加入PEG400混匀;在搅拌下加入上述混合液中;再加0.1%针用活性炭,60℃搅拌15min,脱炭过滤;滤液密闭加热100℃30min,冷却至室温备用,得混合液A;
(3)一边搅拌一边将基因重组人溶菌酶加入占配方量10%的经活性炭处理过的灭菌注射用水,充分溶解,得溶液B。
(4)一边搅拌一边将溶液B以细流加于混合液A中,充分溶解,得溶液C。
(5)用0.1mol的NaOH溶液调节溶液C的pH值至6.0~7.5。
(6)采用已灭菌的0.45μm串联0.22μm微孔滤膜将溶液C过滤除菌。于无菌灌封机中灌封于无菌容器中;灯检、检漏、印字、包装出厂。
试验例1:
基因重组抗病毒人溶菌酶-抗体结合物的抗体结合效价检测。
分别选择HPV6型、HPV11型、HPV16型、HPV18型、HPV31型、HPV33型、HPV45型、HPV52型、HPV53型、HPV58型等型的E6蛋白作为检测抗原,用“ELISA”方法(酶联免疫法)检测所制得的基因重组人溶菌酶-抗体结合物的抗体效价,结果如下表所示:
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注:测试样本中的基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物抗体溶液浓度均为1mg/mL。
从以上检测结果可看出,所制备的基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物对相对应的人乳头瘤病毒(HPV)E6蛋白抗原都有很高的抗体结合效价。
试验例2:
基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物对相对应的人乳头瘤病毒(HPV)E7蛋白的抗体结合效价检测。
分别选择HPV6型、HPV11型、HPV16型、HPV18型、HPV31型、HPV33型、HPV45型、HPV52型、HPV53型、HPV58型等型的E7蛋白作为检测抗原,用“ELISA”方法(酶联免疫法)检测所制得的基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物的抗体效价,结果如下表所示:
注:测试样本中的基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物抗体溶液浓度均为1mg/mL。
从以上检测结果可看出,所制备的基因重组抗病毒人溶菌酶-抗HPV-E6/E7小分子抗体Fab结合物对相对应的人乳头瘤病毒(HPV)E7蛋白抗原都有很高的抗体结合效价。
以上实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改和替换,均属于本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
本发明所包含的信息,在未脱离下述权利要求的精神和保护范围下,本发明各种偏离精确的描述,对于与本发明相关的本领域技术人员来说是显而易见的。本发明并不认为限制在所定义的程序、性质或组成的范围内,因为优选的实施例和其他描述只用于说明目前提供发明的特定方面。对于在化学、生物化学或相关领域的技术人员来说,实现本发明于各种修改的描述模式,都应属于本发明所附权利要求的保护范围内。
应当理解的是,对本领域普通技术人员来说,可以根据上述说明加以改进或变换,所有这些改进或变换都应属于本发明所附权利要求的保护范围之内。
序列表
<110> 深圳市雅臣智能生物工程有限公司
<120> 一种人溶菌酶结合物及其制备方法和应用
<140> 2019105947814
<141> 2019-07-03
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 130
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly
1 5 10 15
Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys Leu Ala
20 25 30
Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn Ala Gly
35 40 45
Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp
50 55 60
Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser
65 70 75 80
Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala Cys Ala
85 90 95
Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val Ala Trp
100 105 110
Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln Gly Cys
115 120 125
Gly Val
130
Claims (1)
1.一种人溶菌酶结合物在制备用于防治人乳头瘤病毒感染药物中的应用;
所述药物以人溶菌酶结合物为唯一活性成分;
所述人溶菌酶结合物包括相互偶联的人溶菌酶与抗体,所述人溶菌酶为基因重组表达的产物;
所述人溶菌酶的氨基酸序列为:
Lys-Val-Phe-Glu-Arg-Cys-Glu-Leu-Ala-Arg-Thr-Leu-Lys-Arg-Leu-Gly-Met-Asp-Gly-Tyr-Arg-Gly-Ile-Ser-Leu-Ala-Asn-Trp-Met-Cys-Leu-Ala-Lys-Trp-Glu-Ser-Gly-Tyr-Asn-Thr-Arg-Ala-Thr-Asn-Tyr-Asn-Ala-Gly-Asp-Arg-Ser-Thr-Asp-Tyr-Gly-Ile-Phe-Gln-Ile-Asn-Ser-Arg-Tyr-Trp-Cys-Asn-Asp-Gly-Lys-Thr-Pro-Gly-Ala-Val-Asn-Ala-Cys-His-Leu-Ser-Cys-Ser-Ala-Leu-Leu-Gln-Asp-Asn-Ile-Ala-Asp-Ala-Val-Ala-Cys-Ala-Lys-Arg-Val-Val-Arg-Asp-Pro-Gln-Gly-Ile-Arg-Ala-Trp-Val-Ala-Trp-Arg-Asn-Arg-Cys-Gln-Asn-Arg-Asp-Val-Arg-Gln-Tyr-Val-Gln-Gly-Cys-Gly-Val;
所述抗体为抗HPV-E6/E7-Fab;
所述人溶菌酶结合物的制备方法,包括如下步骤:
S1、体外表达人溶菌酶:将人溶菌酶基因克隆到表达系统,表达人溶菌酶基因以生产基因重组人溶菌酶;
S2、连接抗体:将所述人溶菌酶与抗体偶联,得到所述人溶菌酶结合物;
S201、用SPDP分别处理人溶菌酶和抗体,收集处理后的人溶菌酶蛋白液和抗体蛋白液;
S202、用DTT处理所述人溶菌酶蛋白液,收集还原的人溶菌酶蛋白液;
S203、将所述还原的人溶菌酶蛋白液和所述抗体蛋白液混合,偶联反应得到所述人溶菌酶结合物。
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US5433955A (en) * | 1989-01-23 | 1995-07-18 | Akzo N.V. | Site specific in vivo activation of therapeutic drugs |
CN1927888A (zh) * | 2006-09-30 | 2007-03-14 | 大连帝恩生物工程有限公司 | Glp-1及其类似物与人溶菌酶融合的重组蛋白及其用途 |
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FR2573627A1 (fr) * | 1984-11-29 | 1986-05-30 | Entremont Sa | Procede pour la fabrication de produits laitiers au moyen d'anticorps |
US5433955A (en) * | 1989-01-23 | 1995-07-18 | Akzo N.V. | Site specific in vivo activation of therapeutic drugs |
CN1927888A (zh) * | 2006-09-30 | 2007-03-14 | 大连帝恩生物工程有限公司 | Glp-1及其类似物与人溶菌酶融合的重组蛋白及其用途 |
CN107847593A (zh) * | 2015-04-20 | 2018-03-27 | 美侬米克国际有限公司 | 治疗抗体及其用途 |
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